Top-Down Analysis with Modified or Unnatural Amino Acids

Byos accommodates two solutions to analyze Top-down MS data of proteins, which are the peptide and intact modules. This article shows the configuration process in the presence of modified or unnatural amino acids for each of these modules.

Peptide Module

General settings for Top-down analysis using Byonic including the modification settings are described here. Byonic can support a limited number of unnatural amino acids residues by redefining one-letter amino acid abbreviations using fixed modifications. Starting with version 2.9.77, Byonic accepts B, Z, U, O, J, and X in FASTA protein databases. The non-standard amino acid masses are:

For example, by placing a fixed modification of +13.04768 on J, the user can make J (in a FASTA database)have a mass of 113.04768 Da, which is correct for hydroxyproline. Byonic does, however, use amino acid sequence to predict peak intensity, so this fixed modification on J will not give the same scores as a +15.9949 variable modification on P. Variable modifications can be applied on fixed modified peptides. This can be set in the custom modification box (see below).

Intact module

General settings for Top-down analysis using the Byos Intact module are described here. In contrast to the peptide module, the intact module focuses on confirming protein sequences via intact mass deconvolution and fragmentation spectra analysis. This module treats modified or unnatural amino acids similarly, emphasizing sequence confirmation rather than positional identification along the peptide backbone.
Defining sequence building blocks is done at the Sequences and masses section of the project creation window by inputting a chemical formula. The average mass is calculated automatically. The masses of the chemical elements can be changed here as well. Below is an example for oxidized Methionine and Hydroxyproline: